A tug-of-war concept has been proposed to explain how a chemotactic stromal derived factor-1 (SDF-1) gradient between bone marrow (BM) and peripheral blood (PB) determines whether cells will be released and mobilized from BM into PB or home back from PB to the BM microenvironment. This concept, however, needs reappraisal because plasma SDF-1 levels do not consistently correlate with mobilization of hematopoietic stem/progenitor cells (HSPCs). At the same time, it is becoming clear that SDF-1 does not play an exclusive role in the homing of HSPCs into BM, implying the existence of additional factors. New data from our laboratory and supported by recent observations from other researchers, identify a potentially important role for the bioactive sphingolipids sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P) in trafficking of HSPCs. S1P and C1P are i) strong chemoattractants for HSPCs, ii) resistant to proteolytic enzymes, and iii) exhibit markedly increased levels in PB during mobilization and in BM after myeloablative conditioning for transplantation. Based on these data, we propose three interrelated aims to reappraise the role of SDF-1 in stem cell trafficking and address our central hypothesis that the bioactive lipids S1P and C1P play an important and presently unappreciated role in homing and mobilization of HSPCs. In addition to clinical studies, our approach will exploit small molecule- and genetics-based strategies in mouse models. Specific Aim 1. Bioactive lipids S1P and C1P as executors of HSPC mobilization. Our recently published data support a pivotal role for the S1P- S1P receptor type 1 (S1P1) axis in egress of HSPCs from BM into PB. We will investigate the impact of mobilization on the distribution of S1P between blood compartments and determine whether the level of S1P in PB correlates with mobilization efficacy in patients. In parallel, we will address the role of C1P and investigate whether one or both bioactive lipids (which are stored in circulating red blood cells) are responsible for mobilization of HSPCs in patients with hemolytic syndromes. Finally, we will mechanistically define which mobilization steps are promoted by S1P and C1P. Specific Aim 2. Bioactive lipids as a novel homing factors for HSPCs. Myeloablative conditioning for transplantation induces a proteolytic microenvironment in BM leading to a decrease in chemotactically active SDF-1. We have found that BM levels of S1P and C1P are increased during conditioning and that antagonism of S1P1 impairs engraftment of HSPCs. Since these lipids are strong HSPC chemoattractants for HSPCs, we will focus on their potential role in homing of HSPCs and elucidate the molecular and cellular basis for this phenomenon. Specific Aim 3. Develop strategies to improve mobilization and homing of hematopoietic stem cells by modulating S1P and C1P signaling. Based on our preliminary data we propose to develop new strategies that will improve both mobilization and homing of HSPCs. These strategies will be tested first in mice and subsequently validated in chimeric human HSPCs/immunodeficient mouse xenotransplant models.